Next generation sequencing-guided anti-infective therapy of large liver abscess due to Edwardsiella tarda: a case report

Introduction

Liver abscess is a rare but potentially fatal disease with mortality rates of 2–12% [1], [2]. Prompt identification of the causative pathogen is very important for effectively anti-infective treatment. However, determining the etiology of liver abscess is still challenging [3]. Conventional blood culture remains routinely used in clinics, but it is time-consuming and pathogen-limited, with a positive rate only 2–25% for liver abscess [4], [5]. Abscess puncture culture has a higher positivity rate, but the invasive biopsy for liver abscess would lead to increased complications and add significant cost owing to prolonged hospitalization [5]. Therefore, an urgent need exists for rapid, sensitive, and minimal-invasive diagnostic method to inform anti-infective therapy in patients with liver abscess. We present a case with large liver abscess due to Edwardsiella tarda (E. tarda) that was minimal-invasively cured with the aid of a novel plasma-based next-generation sequencing (NGS) assay for accurate detection of the pathogenic bacterial genome.

Case presentation

A 65-year-old male presented at an outside hospital with a 2-week fever, exhaustion, poor appetite, and right upper abdominal distending pain. Initial blood culture was negative. Abdominal ultrasonography revealed a well-defined hypoechoic lesion of the hepatic left lobe measuring 7.0×6.5 cm in size, and a provisional diagnosis of liver abscess was made. The patient’s history was notable for brucellosis about 2 years earlier, and a long history of alcohol abuse. He did not have any history of chronic illnesses such as liver disease, diabetes mellitus, tuberculosis, hypertension, heart disease or cancers. Given the history of brucellosis with similar manifestations, he was treated empirically with intravenous rifamycin, without improvement. Thereafter, he was transferred to our hospital for further diagnosis and treatment.

On the day of admission, the patient had a temperature of 38.2°C, pulse rate of 102/min, blood pressure of 90/60 mm Hg and respiratory rate of 25/min. He had anorexia and felt weak, but denied vomiting, diarrhea or constipation. Cough or respiratory symptoms were absent. The patient had right upper quadrant abdominal tenderness with no distension or rigidity. Laboratory examinations revealed a white blood cell count of 10,000/mm³ (normal, 3500–9500/mm³) with neutrophils 77.7% (normal, 50–70%), a platelet count of 395,000/mm³ (normal, 100,000–350,000/mm³); C-reactive protein level was 36.8 mg/L (normal, <8.20 mg/L) and procalcitonin was 4.63 ng/mL (normal, <0.5 ng/mL). Liver and renal function tests were within reference ranges. Serology for hepatitis B and C was negative. Urine, stool, sputum, and two blood samples on the day of admission were collected for culture, but failed grow any microorganisms. Blood film for malarial parasites was negative. Chest radiograph was normal. Magnetic resonance imaging (MRI) scan with gadolinium enhancement of the abdomen revealed a rim-enhancing abscess lesion measuring 7.3×6.6×6.2 cm in the left hepatic lobe (Figure 1A). Ultrasound guided percutaneous drainage of liver abscesses and subsequent pus culture was intended to perform, but the patient declined the invasive manipulation.

Figure 1:

Magnetic resonance imaging (MRI) of the abdomen on admission and 1-, 3-, and 6-month after anti-infective therapy.

(A) T1-weighted MRI with gadolinium enhancement on admission showed a large, fluid-containing lesion with rim enhancement in the left lobe of liver, indicated by an arrow. (B) MRI with enhancement on 1-month after therapy showed a decrease in size of the liver abscess cavity. (C) T2-weighted MRI on 3-month after therapy showed a further decrease in size of the liver abscess. (D) MRI with enhancement on 6-month after therapy showed the abscess disappeared completely.

Due to the uncertainty of the etiology of this illness, a NGS-based plasma assay was run which identified E. tarda within 24 h. Volume of 10 mL of venous blood was collected in Streck cell-free DNA (cfDNA) tubes and cfDNA was extracted using QIAamp circulating nucleic acid kit (QIAGEN, Valencia, CA, USA). High throughput sequencing (MedcareDx Bio-Tech, Shanghai, China) was conducted on the cfDNA sample by Illumina NextSeq550Dx platform. Twenty-five million reads from the sample were acquired on the sequencer. Low-quality base calls, adapter sequences were trimmed off from the raw reads, and duplicate were removed. High level (Q30) reads were aligned to the hg19 human reference sequence, then the unmapped reads were aligned to the bacterial, viral, fungal and parasite databases. Finally, the NGS assay detected high levels of microbial DNA with reads that aligned along the entirety of the E. tarda genome (Figure 2). Further 16S rRNA gene amplification using specified primers (forward primer: GAGTTTGMTCCTGGCTCAG; reverse primer: CTGCTGGCACGGAGTTAG) described by Li et al. [6], and sequencing analysis showed the PCR-amplified product was identical to the sequence from E. tarda.

Figure 2:

Average alignment coverage over the entire Edwardsiella tarda genome.

Based on this result, the patient was treated with ceftriaxone (2 g/day) as a single agent [7]. By the third day following the anti-infection therapy, the patient defervesced and clinically improved. By the seventh day repeat laboratory examinations revealed a white blood cell count of 5850/mm³ with neutrophils 62.6%, and normal C-reactive protein (7.19 mg/L) and procalcitonin (0.39 ng/mL). A second NGS assay of the blood sample obtained on day 10 following anti-infection therapy, showed that the pathogenic E. tarda DNA was undetectable. The patient completed a 2-week course of anti-infective treatment, and eventually discharged on the 18^th day, without any discomforts. He has since been doing well and remains free of disease recurrence during 1 year of follow-up. Follow-up MRI of the abdomen after 1 month and 3 months showed a gradually decrease in size of the liver abscess (Figure 1B,C), and the MRI scan after 6 months showed that the abscess disappeared completely (Figure 1D).

Informed consent was obtained from the patient included in this study. The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ Institutional Review Board.

Discussion

To our knowledge, this is the first reported case in which a NGS-based plasma cfDNA assay was used to detect the genome of E. tarda in a patient with liver abscess. Edwardsiella tarda is a facultatively anaerobic gram-negative bacillus belonging to the Enterobacteriaceae family. For humans, E. tarda is a rare but life-threatening opportunistic pathogen, and the overall mortality rate has been reported to be up to 50% [8]. Most cases of human infection present as gastroenteritis, but extraintestinal infections, including liver abscess, septicemia, meningitis, cholecystitis, osteomyelitis, endocarditis, empyema, peritonitis, and wound infection, have also been reported less frequently but more lethally [7], [9], [10]. Clinical manifestations of E. tarda infection are nonspecific and the diagnosis in clinical practice relies on culture-based pathogen identification techniques [11]. However, the standard culturing method has obvious limitations such as insufficient sensitivity and being time-consuming, which may contribute to a high case fatality rate [12]. Therefore there is a need for more effective diagnostic test that can overcome these limitations to aid in guiding therapy.

NGS is a powerful tool with potential to revolutionize infectious disease diagnosis pattern and offers several advantages over existing pathogen identification techniques [13]. Firstly, the platform has extreme sensitivity and completely open-ended nature, allowing for hypothesis-free detection of any microorganism. Secondly, NGS is high-throughput and returns results within a clinically actionable timeframe, which provides clinicians with more actionable information. Thirdly, the use of cfDNA in patient peripheral blood provides easily accessible specimens to diagnose microorganism infections. Finally, the molecular diagnostic technique can quantitate pathogen DNA, indicating it could be a useful tool in monitoring infection course. For the case we presented here, E. tarda was identified as pathogenic bacteria in the first detection, and the pathogen DNA copy reduced to undetectable in the second detection after effectively treatment. Similarly, in a patient with neurocysticercosis owing to Taenia solium, the tapeworm DNA index dropped considerably in the secondary NGS test after effectively antiparasitic treatment [14]. These data suggested that kinetic analysis of pathogen DNA may provide useful clinical insights in patients with microorganism infection. Further studies are needed to determine whether or not the kinetics of pathogenic DNA detected by NGS can be used for efficacy assessment.

Many treatment strategies have been proposed for liver abscess, with small abscess (<3–5 cm) being treated with antibiotics alone, and large abscess with percutaneous drainage and/or surgical therapy plus antibiotics [4], [15]. For our case with a 7.3-cm liver abscess, percutaneous transhepatic drainage and antibiotics are generally selected as first-line therapy. But due to the refusal of this patient to invasive operation, we had few ways to timely diagnose and treat the disease. However, with the aid of the NGS-based pathogenic diagnosis, the patient received timely precision antibiotic and completely recovered from the disease. The present report indicates that antibiotic alone is an alternative to first-line treatment for large liver abscess, on condition that the pathogen can be defined, and thus emphasizes the importance of accurate pathogenic diagnosis. In this respect, NGS technology may pave the way to treatment pattern conversion for large liver abscess from invasive therapy to non-invasive therapy.

In conclusion, the plasma NGS test can sensitively and minimal-invasively detect pathogens within a clinically actionable timeframe, which would aid in the timely precision therapy for patients with microbial infection. Antibiotic alone is an alternative to first-line treatment for large liver abscess with the aid of NGS for accurate detection of the bacterial genome.